Morphological Variation and Discriminating Traits of Kersting’s Groundnut Accessions

Gilles Y. Chodaton University of Abomey Faculty of Agricultural Sciences: Universite d'Abomey-Calavi Faculte des Sciences Agronomiques Eric Etchikinto Agoyi (  [email protected] ) University of Abomey Faculty of Agricultural Sciences: Universite d'Abomey-Calavi Faculte des Sciences Agronomiques Thomas A. Houndété Institut National des Recherches Agronomiques Konoutan M. Kafoutchoni Universite d'Abomey-Calavi Faculte des Sciences et Techniques Hospice S. Sossou University of Abomey Faculty of Agricultural Sciences: Universite d'Abomey-Calavi Faculte des Sciences Agronomiques Fréjus A.K. Sodédji Korean Advanced Institute of Science: Korea Advanced Institute of Science and Technology Sergino Ayi University of Abomey Faculty of Agricultural Sciences: Universite d'Abomey-Calavi Faculte des Sciences Agronomiques Symphorien Agbahoungba University of Abomey Faculty of Agricultural Sciences: Universite d'Abomey-Calavi Faculte des Sciences Agronomiques Flora J. Chadare UNA: Universite Nationale d'Agriculture Appolinaire Adandonon UNA: Universite Nationale d'Agriculture Raymond Vodouhè Bioversity International: Alliance of Bioversity International and International Center for Tropical Agriculture Achille E. Assogbadjo University of Abomey Faculty of Agricultural Sciences: Universite d'Abomey-Calavi Faculte des Sciences Agronomiques Brice A. Sinsin University of Abomey Faculty of Agricultural Sciences: Universite d'Abomey-Calavi Faculte des Sciences Agronomiques

Research Article

Keywords: Descriptors, diversity, Kersting’s groundnut, geocarpum, orphan crops

Posted Date: August 9th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-671485/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 Morphological variation and discriminating traits of Kersting’s groundnut accessions

2 Gilles Y. Chodaton1,2, Eric E. Agoyi1*, Thomas A. Houndété4, Konoutan M. Kafoutchoni1, Hospice S. 3 Sossou1, Fréjus A.K. Sodédji1, Sergino Ayi1, Symphorien Agbahoungba1, Flora J. Chadare5, Appolinaire 4 Adandonon2, Raymond Vodouhè3, Achille E. Assogbadjo1, Brice A. Sinsin1 5 6 1Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied Ecology, Faculty of 7 Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin; 8 2School of Crop and Seeds Production and Management, National University of Agriculture; BP 43 Kétou, Tel: 9 (229) 21 00 29 98 10 3Benin-Bioversity International ; Tel: 21 35 01 88 / 21 35 06 00, 11 4Centre de Recherches Agricoles Coton et Fibres, Institut National des Recherches Agricoles du Bénin, 01 BP 12 175, Cotonou, Bénin 13 5 School of Sciences and Techniques for Preservation and Processing of Agricultural products, National 14 University of Agriculture, P. O. Box 114, Sakété, Benin. 15 *Corresponding author: [email protected] 16 phone: (+229) 97 98 97 45/ 95 19 90 55, 17 ORCID-ID: https://orcid.org/0000-0003-1659-6534 18

19 Authors

20 Gilles Y. Chodaton: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied Ecology, 21 Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin. 22 ([email protected]) 23 Eric Etchikinto Agoyi*: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied 24 Ecology, Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin. 25 (*Corresponding author: [email protected]; ORCID-ID: https://orcid.org/0000-0003-1659-6534) 26 Thomas A. Houndété: Centre de Recherches Agricoles Coton et Fibres, Institut National des Recherches 27 Agricoles du Bénin, 01 BP 175, Cotonou, Bénin. ([email protected]) 28 Konoutan M. Kafoutchoni: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied 29 Ecology, Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin. 30 ([email protected]; https://orcid.org/0000-0002-9153-4272) 31 Hospice S. Sossou: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied Ecology, 32 Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin. 33 ([email protected]) 34 Fréjus Ariel Kpèdétin Sodédji: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of 35 Applied Ecology, Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, 36 Benin. ([email protected] http://orcid.org/0000-0003-3450-3346) 37 Sergino Ayi: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied Ecology, 38 Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin. 39 ([email protected]) 40 Symphorien Agbahoungba: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied 41 Ecology, Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin. 42 ([email protected])

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43 Flora J. Chadare: School of Sciences and Techniques for Preservation and Processing of Agricultural products, 44 National University of Agriculture, P. O. Box 114, Sakété, Benin. ([email protected]) 45 Appolinaire Adandonon: School of Crop and Seeds Production and Management, National University of 46 Agriculture; BP 43 Kétou, Tel: (229) 21 00 29 98. ([email protected]) 47 Raymond Vodouhè: Benin-Bioversity-International; Tel: 21 35 01 88 / 21 35 06 00. ([email protected]) 48 Achille E. Assogbadjo: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied 49 Ecology, Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin. 50 ([email protected]) 51 Brice A. Sinsin: Non-Timber Forest Products and Orphan Crop Species Unit, Laboratory of Applied Ecology, 52 Faculty of Agronomic Sciences, University of Abomey-Calavi, 01 BP 526, Tri postal, Cotonou, Benin. 53 ([email protected] )

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54 Abstract

55 Kersting’s groundnut [Macrotyloma geocarpum (Harms) Maréchal & Baudet] (KG) is a nutritious, subterranean 56 grain legume in West and Central Africa. Only limited information is available on the morphological traits that 57 can discriminate accessions; without such information, appropriate breeding strategies cannot be devised. This 58 study aimed to identify discriminating traits and assess the diversity among accessions of Kersting’s groundnut. 59 Eighty-one KG accessions from Benin and Burkina Faso were evaluated based on 29 qualitative and quantitative 60 traits. An experiment was conducted using an Alpha lattice design with three replications. Standardized Shannon- 61 Weaver index (H') and descriptive statistics were calculated for qualitative traits. Pearson correlation coefficients, 62 stepwise discriminant analysis, principal component analysis, cluster analysis and canonical discriminant analysis 63 were conducted. Results showed that accessions varied greatly based on growth habit (H'= 0.68), flower color (H' 64 = 0.50), seed-eye shape (H' = 0.47), and stem pigmentation (H' = 0.41). Eight quantitative traits, viz., seed width, 65 seed thickness, number of branches per , petiole length, days to 50% flowering, number of seeds per pod, pod 66 width, and pod length, were found to significantly discriminate the accessions. Accessions were grouped into three 67 clusters based on quantitative traits. Cluster 1 had accessions with late flowering and good vegetative growth, 68 Cluster 2 contained accessions with high germination percentage and Cluster 3 had accessions with high yield 69 performance. Seed length varied greatly among accessions, thus indicating the potential for improving yield via 70 seed size. 71 Keywords: Descriptors, diversity, Kersting’s groundnut, Macrotyloma geocarpum, orphan crops. 72

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78 1. Introduction

79 Orphan crops, also known as forgotten or abandoned crops, traditional or underdeveloped crops

80 (Padulosi et al., 2013), are crop species that have received only limited attention from

81 researchers. However, most orphan crops are highly nutritious, climate resilient (Mabhaudhi et

82 al., 2019), and resistant to commonly occurring crop diseases (Andrew et al., 2009). Thus,

83 orphan crops can potentially contribute toward food security and nutritional security, and should

84 receive more research attention.

85 Kersting’s groundnut [Macrotyloma geocarpum (Harms) Maréchal & Baudet)] is a

86 multipurpose legume crop that is widely grown in West Africa and Central Africa (Adu-Gyamfi

87 et al., 2011; Abiola and Oyetayo, 2015). It is reportedly an orphan and underutilized crop

88 species (Adu-Gyamfi et al., 2012; Dansi et al., 2012) that thrives well in semi-arid zones with

89 an annual rainfall of <600 mm (Achigan Dako and Vodouhè, 2006). Kersting’s groundnut has

90 high nutritional value, as it contains 21.3 g protein per 100 g of grain (Ajayi and Oyetayo, 2009).

91 The seed is a rich source of crude protein, with high levels of essential amino acids, such as

92 phenylalanine (3.2/100 g), histidine (2.1/100 g), lysine and methionine (Ajayi and Oyetayo,

93 2009). Seeds have high contents (Leung et al., 1968). According to Adazebra (2013),

94 Kersting’s groundnut is one of the less-known leguminous crops, but it contributes significantly

95 toward rural nutrition, livelihoods and sustainable development. Highly appreciated in urban

96 areas of Benin, the crop has a high market value; it is the most expensive grain legume in West

97 Africa, selling at US$ 5-7 per kilogram (Agoyi et al., 2019). Despite its nutritional and

98 economic values, Kersting’s groundnut cultivation continues to decline in West African

99 countries because of constraints, such as low yield, non-availability of improved varieties, poor

100 storage ability of the grains, and high labor requirements for production (Ayenan and Ezin,

101 2016). It is not a priority crop for governments and researchers (Dansi et al., 2012; Adazebra,

102 2013; Assogba et al., 2015).

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104 Agro-morphological characterization is a key step in assessing genetic diversity to classify

105 germplasm of cultivated (Boyé et al., 2016; Radhouane, 2004). To do so, researchers use

106 descriptors. However, descriptors for Kersting’s groundnut have not been described, unlike

107 Bambara groundnut ( (L.) Verdc.) (IPGRI et al., 2000), (Arachis

108 hypogaea L.) (IBPGR and ICRISAT, 1992) and pigeonpea (Cajanus cajan (L.) Millsp.)

109 (IBPGR and ICRISAT, 1981). Only a few studies have focused on studying morphological

110 variation in Kersting’s groundnut, most of which evaluated either few number of accessions or

111 accessions from only one country. Assogba et al. (2015) and Akohoue et al. (2019) in Benin

112 and Adu-Gyamfi et al. (2012) in Ghana and Bayorbor et al. (2010) in Nigeria reported

113 significant variation for various traits among accessions.

114 The present study aimed at filling the above-mentioned gaps by i) assessing diversity among

115 accessions in a regional germplasm collection, obtained from Benin and Burkina Faso, and ii)

116 identifying discriminating traits that could be included in a list of descriptors to be used for

117 morphological characterization of Kersting’s groundnut.

118 2. Materials and methods

119 2.1. Study area

120 The study was carried out at the Regional Center of Agricultural Research (CRA-CF) in Djidja,

121 village of Djegbatin (7°19'04.362'' N and 1°54'58.914'' E). The climate of Djidja is sub-

122 equatorial and the rainfall is generally bimodal but can also be unimodal. The soils are ferralitic,

123 ferruginous and hydromorphic. Rainfall, temperature, sunshine, and relative humidity during

124 the period of experimentation are presented in Table 1.

125 Table 1. Monthly average climatic data recorded on study site during experiment.

126 2.2. Plant material and experimental design

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127 Genetic material consisted of a collection of 81 accessions, of which 70 were from Benin and

128 11 from Burkina Faso (Table 2). Planting was done on 23 August 2018. The experiment was

129 conducted using an Alpha lattice design, with 9 plots per block × 9 blocks and three replications.

130 Each plot consisted of three rows, each 4.5 m in length. The rows were spaced 0.75 m apart.

131 Plant-to-plant spacing was 0.30 m, giving a plant population of 44500 plants per hectare.

132 Distance between plots was 1 m. One seed was sown per hill at a depth of 5 cm. No fertilizer

133 was applied and weeding was done manually 3 weeks, 7 weeks and 12 weeks after sowing.

134 Table 2. Name, seed color and origin of Kersting’s groundnut accessions used in this study.

135 2.3. Data collection and analysis

136 The quantitative traits evaluated were: germination percentage, number of leaves per plant,

137 number of flowers per plant, number of pods per plant, yield, number of branches per plant,

138 days to 50% flowering, 100-seed weight, seed length, seed width, seed thickness, leaf length,

139 leaf width, petiole length, pod length, pod width, and number of seeds per pod. In addition, data

140 were collected on 12 qualitative traits (Table 3).

141 Table 3. Qualitative morphological traits evaluated

142 The traits measured were adapted from the lists of descriptors of closely related and similar

143 subterranean legume species, such as Bambara groundnut (IPGRI et al. 2000) and peanut

144 (IBPGR and ICRISAT 1992). Observations were made on 20 randomly selected plants within

145 each plot. Standardized Shannon-Weaver index (H') was calculated for the qualitative traits

146 (Ghimire et al., 2018; Yadav et al., 2018). For the quantitative traits, descriptive statistics (mean,

147 standard deviation, minimum, maximum, coefficient of variation) were calculated.

148 Although studies have used the standardized Shannon-Weaver diversity index (H’) for both

149 quantitative and qualitative traits (Ghimire et al. 2018; Yadav et al. 2018), we used coefficient

150 of variation for quantitative traits. In fact, the calculation of H’ requires recording continuous

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151 data into a set of discrete categories (i.e., binning). However, evidence shows that binning often

152 results in loss of information because of reduction in data points (Anderson et al., 2008;

153 Sengupta and Sil, 2020). Besides, the choice of the cut-off point and the amplitude of the

154 defined phenotypic classes are totally arbitrary and left to the discretion of the researcher,

155 leading to difficulties in comparing results across studies.

156 Coefficient of variation (CV) (%) was computed to assess the level of phenotypic variation in

157 quantitative traits, as follows:

158 CV (%) (Abdi, 2010) S 159 where s = the standard deviation and= 푥=̅ the× 100mean.

160 For qualitative traits, H' was calculated푥̅ in Microsoft Excel based on phenotypic frequencies of

161 each trait to evaluate the variability among accessions using the following formula:

162 H' (Yadav et al.2018) [Σ (n / N) × {log2 (n / N) ∗ (− 1)}] log2k 163 where H' is the standardized= Shannon-Weaver diversity index, k is the number of phenotypic

164 classes for a given qualitative trait, n is the frequency of the phenotypic class for each trait and

165 N is the total number of observations.

166 Analysis of variance (ANOVA) using a linear mixed model for Alpha lattice designs was used

167 to test for differences among accessions for quantitative traits. The linear mixed model used

168 was as follows (genotypes were considered fixed effects and replications and blocks random

169 effects):

170 (Asharaf et al. 2013)

푖푗푘 푖 푗 푘 푖푗푘 171 where = value of푌 the= observed 휇 + 퐺 + quantitative 푅 + 퐵 + trait; 휀 = population mean; = effect of the ith

푖푗푘 푖 172 accession푌 ; = effect of the jth replicate (superblock); 휇 = effect of the k 퐺th incomplete block

푗 푘 173 within the j푅th replicate; and = experimental error. 퐵

휀푖푗푘

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174 Pearson's correlation was used to examine the relationship between yield and other quantitative

175 traits. Further, quantitative trait data were subjected to stepwise discriminant analysis to

176 determine the traits that best discriminated the accessions. Canonical discriminant analysis was

177 performed to describe relationship between seed and flower color based on discriminating traits.

178 Principal component analysis (PCA) was performed to determine the patterns of agro-

179 morphological variation. Hierarchical classification was done to group the accessions.

180 Thereafter, descriptive statistics and analysis of variance (ANOVA) were used to describe the

181 clusters. All analyses were performed in R software 3.5.2. (R Core Team, 2019).

182 3. Results and discussion

183 The standardized Shannon Weaver diversity index (Hʹ) values ranged from 0.16 to 0.68 (Table

184 4). There was a high level of phenotypic variation among accessions for plant growth habit

185 (H’=0.68) and flower color (H’=0.50). Moderate variation was observed for seed-eye shape,

186 easy pod detachment, stem pigmentation and seed coat color. Pod color, terminal leaflet shape,

187 terminal leaflet color, pod shape and pod texture exhibited a relatively low level of variation

188 (Table 4). Growth habit and flower color could be used as key qualitative descriptors for

189 Kersting’s groundnut.

190 A large majority (73%) of the accessions in the collection exhibited prostrate growth habit. This

191 could be used to inform breeding strategies. For instance, Ndiang et al. (2012) reported prostrate

192 growth habit as a good yield predictor in Bambara groundnut, a subterranean legume crop

193 similar to Kersting’s groundnut. In addition, all accessions from Burkina Faso had elongated

194 seed (Table 4). Big seed could be used as a selection criterion to improve yield.

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196 Table 4. Phenotypic variability observed in accessions based on the calculation of the Standardized 197 Shannon-Weaver index (H’) 198

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199 Most accessions (88.88%) had greenish-white flowers (Figure 1a) and the rest of them (11.12%)

200 had purple-tinted white flowers (Figure 1b). Similarly, 95.06% of the accessions had white pods

201 (Figure 1c) and only 4.94% had white pods with purple tint (Figure 1d). Three colors of seed

202 coat were observed among the germplasm collection. Most accessions (90.12%) had cream seed

203 coat (Figure 1e), while 8.64% had black seed coat (Figure 1f) and 1.24% had red-brown seeds

204 (Figure 1g). This result could be explained by the fact that black-seeded and the brown-seeded

205 accessions were rare and were produced by a few households on a small scale (Akohoue et al.

206 2018).

207 Similarly, three variants were recorded for seed-eye shape. A large majority of accessions

208 (85.20%) had triangular seed eyes (Figure 1i), while 7.40% had butterfly-like seed eyes (Figure

209 1h), and 7.40% had irregular seed eyes (Figure 1j).

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211 Figure 1. 1a) greenish-white flowers, Fig 1b) purple-tinted white flowers, Fig 1c) white 212 pod Fig 1d) purple tinted-white, Fig 1e) Cream coat seed, Fig 1f) Black coat seed, Fig 1g) 213 brown coat seed, Fig 1h) butterfly shape eyes, Fig 1i) triangular eyes, Fig 1j) irregular 214 eyes. 215

216 Grain yield and yield components, i.e., number of pods, number of flowers and number of

217 branches per plant showed relatively high coefficients of variation (34.78% - 50.19%) (Table

218 5). This is consistent with findings on bambara groundnut (Boyé et al. 2016), cowpea (Gbaguidi

219 et al. 2015) and Kersting’s groundnut (Assogba et al. 2015), where high and significant

220 coefficients of variation were observed for number of flowers per plant. The high CV values

221 are indicative of the existence of substantial diversity among accessions, offering opportunities

222 for improving the trait(s) studied. However, this study showed low variation for seed size (CV=

223 5.06 %, 4.63 %, and 5.37 % for seed length, width and thickness, respectively). Seed size is an

224 important trait for Kersting’s groundnut, since the tiny seeds make harvesting difficult and cause

225 significant yield loss. In fact, harvesting of Kersting’s groundnut is done by hand-picking pods

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226 and shelling consists of thrashing dry pods. Hand-picking of pods with tiny seeds is difficult

227 and bear high chances of leaving out many pods are high. In addition, tiny seeds lead to

228 increased loss during shelling and winnowing. The importance of seed size in Kersting’s

229 groundnut has been recognized in previous studies. For instance, Amujoyegbe et al. (2007)

230 reported small seed size to be one of the major causes of decline of Kersting’s groundnut

231 production in Nigeria. Breeding for bigger seeds, in addition to improving yield, would relieve

232 women from drudgery while hand-picking and shelling Kersting’s groundnut pods. Consistent

233 with Assogba et al. (2015) [100-seed weight, (range =10.70 to 14.71 g)] and Akohoue et al.

234 (2019) [100-seed weight (range = 7.10 to 16.28 g)], the present study showed significant

235 variation (p < 0.001) for 100-seed weight (range = 8.14 to 18.64 g) (Table 5). The slightly bigger

236 seeds observed could be explained by different experimental conditions (climatic and soil

237 conditions), as reported by Khan et al. (2010) that accumulation of reserves in seeds depends

238 on the type of genotypes but also climatic factors. In fact, the present study was conducted on

239 a well-watered fallow in the top Kersting’s groundnut-producing area, known as the food basket

240 of southern Benin. Nevertheless, investigations need to be pursued further, with multi-location

241 trials to fully understand the determinants of yield variation in the crop.

242

243 Table 5. Minimum, maximum, mean and variation in traits of Kersting’s groundnut 244 accessions from Benin and Burkina Faso 245

246 Analysis of variance (ANOVA) performed on quantitative traits showed highly significant

247 differences (p < 0.001) among accessions for seed thickness, percentage of germination, number

248 of flowers per plant, number of days to 50% flowering, seed weight, petiole length and pod

249 length (Table 6). Accessions differed significantly for number of branches per plant, leaflet

250 length (p < 0.01), and leaflet width (P<0.05). Accessions did not vary significantly based on

251 traits such as number of pods per plant, pod width, number of seeds per pod, number of leaves

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252 per plant and grain yield. This difference observed could be explained by genetic variation

253 among the accessions.

254

255 Table 6. ANOVA of the 17 quantitative traits of kersting’s groundnut

256

257 The correlation analysis revealed strong relationships between some of the parameters assessed

258 (Table7). A positive correlation was observed between 100-seed weight and seed length (r =

259 0.68), leaflet length (r=0.38) and petiole length (r=0.42). This result corroborates the

260 observations made by Gbaguidi et al. (2017) on Bambara groundnut. The positive correlation

261 between some of the traits can be exploited for indirect selection. For instance, the positive

262 correlation between number of pods and yield (r=0.59) is an indication that elite plants can be

263 selected based on visual assessment of pod number. On the other hand, low and negative but

264 significant correlation (r= -0.17) was found between days to 50% flowering and number of pods

265 per plant (Table7). These results corroborate results of Assogba et al. (2015) and Yadav et al.

266 (2015).

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268 Table 7. Correlations between agronomic traits for 81 accessions of Kersting’s groundnut 269

270 Stepwise discriminant analysis (SDA) performed on quantitative traits revealed 8 traits, viz.,

271 seed width, seed thickness, number of branches per plant, petiole length, days to 50% flowering,

272 number of seeds per pod, pod width, and pod length, which discriminated the accessions (Table

273 8). These discriminating traits could be used as descriptors for describing Kersting’s groundnut

274 accessions. In fact, being under-researched, Kersting’s groundnut does not have a list of

275 described descriptors to be used for characterizations, unlike many well-studied crops, whose

276 lists of descriptors for morphological traits have been developed and made available by IPGRI,

277 Bioversity International, USDA, ICRISAT or other well-known or international agricultural

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278 research institutes. The eight discriminating traits that were identified constitute a starting point

279 for the establishment of a list of descriptors for the crop.

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281 Table 8. Summary of the stepwise discriminant analysis identifying quantitative traits that 282 differentiated kersting’s groundnut accessions and correlation between discriminating 283 traits and the canonical axes 284

285 Canonical discriminant analysis (CDA), performed to describe seed color of accessions based

286 on discriminating traits, showed two axes that explained 100% of the variation, with the axis 1

287 capturing 96.6% of the variation (Figure 2). Seed thickness, number of branches per plant and

288 days to 50% flowering were correlated with the first axis on the positive side, whereas seed

289 width, petiole length, number of seeds per pod were on the negative side. Thus, axis 1 can be

290 considered indicative of vegetative growth. Seed width, seed thickness, pod length and pod

291 width were correlated with the second axis on the positive side, whereas number of branches

292 per plant and petiole length were correlated with the second axis on the negative side. Most of

293 these traits were related to yield (Figure 2). Overall, black-seeded accessions had wide seed,

294 long pods, a high number of seeds per pod and long petioles. Brown seeds had high pod width,

295 whereas cream-colored seed took more days to reach 50% flowering, and had higher number

296 of branches and thicker seeds (Figure 2).

297

298 Figure 2. Projection of discriminating traits with seed coat color onto the canonical axes 299 1 and 2. 300

301 On their part, accessions with white flowers had thick seeds, whereas accessions with purple

302 flowers had thin seed, and low seed length, leaflet width and petiole length (Figure 3). Thus,

303 white-flower accessions exhibited higher performance for yield components and could be used

304 as donor parents in breeding programs.

305

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306 Figure 3. Boxplots showing relationship between flower color and quantitative traits

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308 In total, significant morphological variation, beyond seed and flower colors, existed among

309 accessions of Kersting’s groundnut. However, the genetic nature of such variation can only be

310 understood if characterization using appropriate molecular marker systems, such as simple

311 sequence repeats (SSRs) or single nucleotide polymorphisms (SNPs) is performed. To date,

312 only one molecular diversity study has been reported using isozymes in this species and no

313 diversity was observed (Pasquet et al., 2002). This state of knowledge needs to be improved

314 and the use of the Next Generation Sequencing Technology may help broaden our knowledge

315 of the genetic diversity in the species.

316 Principal component analysis (PCA) revealed that the first three components had eigenvalues

317 of >1.00 and accounted for 56.4 % of the total variability. The first principal component (PC1),

318 which explained 33.3% of the total variation, was positively associated with seed width, petiole

319 length, leaflet length, germination percentage, pod length, seed length, number of flowers per

320 plant and 100-seed weight; whereas days to 50% flowering was correlated with PC1 on the

321 negative side. The PC1 explained yield traits. The PC2 explained 13.8 % of the total variation

322 and was positively correlated with number of branches per plant, number of leaves per plant

323 and leaflet width. Seed thickness was correlated with PC2 on the negative side (Figure 4).

324

325 Figure 4. PCA biplot of quantitative trait for 81 Kersting's groundnut accessions (Axis 1 326 and Axis 2) 327

328 The PC2 explained vegetative growth. PC3 accounted for 9.3 % of the total variation and was

329 positively correlated with pod width and leaflet width; whereas yield and number of pods per

330 plant were negatively correlated with PC3 (Figure 5). The PCA showed that accessions with a

331 high number of flowers also had long pods, long and tick seeds, long leaflets and high 100-seed

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332 weight. Most of the accessions in that group were from Burkina Faso (BUR3, BUR7, BUR8,

333 BUR9, BUR13, BUR14, BUR15, BUR16 and BUR18) and only three were from Benin (Gbo4,

334 LeAd2 and Zhla2) (Figure 4). Moreover, the accessions that had a high leaflet length also had

335 high number of leaves and branches. Accessions falling into this category were from Benin

336 (Zhla1, Ako and Zke) (Figure 5). In addition, accessions that had high seed thickness also had

337 high leaflet width and high number of pods per plant (Figure 5). Such accessions were Odm2,

338 Agn1, Aso, Kno2, Fol, and Tos, all from Benin.

339

340 Figure 5. PCA biplot of quantitative trait for 81 Kersting's groundnut accessions (Axis 1 341 and 3) 342

343 The UPGMA (unweighted pair group method with arithmetic mean) dendrogram based on

344 discriminating quantitative traits classified the accessions into three clusters (Figure 6).

345

346 Figure 6. Hierarchical clustering of Kersting's groundnut accessions based on quantitative 347 traits. 348

349 The first, second and third clusters contained 68, 3 and 10 accessions, respectively. Cluster 1

350 (C1) was composed of high number of branches per plant (10.03 ± 0.23), high number of leaves

351 per plant (67±1.3), high number of days to 50% flowering (49.26 ± 0.13) and wide leaflet width

352 (49.03±0.21 mm). Accessions belonging to C1 also had low 100-seed weight (12.54 ± 0.09 g),

353 small seed length (8.11 ± 0.03 mm) and low number of flowers per plant (14.7 ± 0.39) (Table

354 9). Overall, C1 was characterized by accessions with high vegetative growth and late flowering.

355 C2 was characterized by a significant (p<0.001) germination percentage (60.47 ± 4.72), high

356 number of flowers per plant (25.8±0.6) and wide petiole length (165.82 ± 2.53 mm), and fewer

357 days to 50% flowering (46.11 ± 0.11). In addition, accessions in C2 had medium number of

358 branches per plant (7.57 ± 1.17) and medium seed length (8.30 ± 0.12 mm) (Table 9) compared

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359 to accessions in clusters 1 and 3. C3 was characterized by high 100-seed weight (15.18 ± 0.29g),

360 high seed length (8.88 ± 0.12 mm), high pod width (7.94± 0.06), long leaflet (69.4 ± 0.71) and

361 wide seed width (6 ± 0.06 mm) but low number of branches per plant (6.56 ± 0.37). Overall,

362 C3 was characterized by accessions with high performance for yield components (Table 9). In

363 Benin, accessions with cream seed coat and eye color (pure cream) are preferred the most.

364 Accessions in C3 showed high performance for yield-related traits, such as seed weight, seed

365 length and seed width. These accessions were Zhla 2, Gbo 4, BUR 3, BUR 7, BUR 8, BUR 9,

366 BUR 14, BUR 16 and BUR 18, all having black or brown seed coat or black-eyed seeds; none

367 of these accessions had pure cream color. Breeding efforts could therefore perform backcross

368 between pure cream accessions and accessions from C3 to obtain improved pure cream varieties

369 with high yield performance.

370

371 Table 9. Mean values and standard errors of discriminating traits in Kersting’s groundnut 372 accessions 373 374 4. Conclusion 375 The evaluation of 81 KG accessions based on the 29 traits revealed high diversity, both for

376 qualitative and quantitative traits. Three diversity groups were identified based on the

377 quantitative traits, clusters were characterized by late flowering, good vegetative growth, high

378 germination percentage and high yield performance. Besides, the study identified seed width,

379 seed thickness, number of branches per plant, petiole length, days to 50% flowering, number of

380 seeds per pod, pod width, and pod length as the quantitative traits that best discriminated the

381 accessions. This could be a starting point for the establishment of a list of descriptors to be

382 measured while studying the crop.

383 Acknowledgement

384 The authors are grateful to NOW-WOTRO for financial support that enabled field experiments.

385 Acknowledgment goes to the Regional Universities Forum for Capacity Building in Agriculture

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386 (RUFORUM) for funding part of the experiments through Carnegie Cooperation of New York.

387 We are grateful to The World Academy of Science (TWAS) for its support. We are grateful to

388 the Benin Institute of Agricultural Research (INRAB) that has provided site and technical

389 support for the experiments.

390 3. Conflict of Interest Statement

391 The authors declare that the research was conducted in the absence of any commercial or

392 financial relationships that could be construed as a potential conflict of interest.

393 4. Authors contributions

394 AEE, SFAK, HS and VR designed the study. CYG, AEE and HS conducted the experiments.

395 CYG and SA actively participated in data collection. KK and SA analysed the data with support

396 from AEE. HAT provided study site and technical guidance. CYG, AEE, KK, HS and SFAK

397 developed the manuscript. VR, AS, AA, CJF, AAE and BS provided guidance throughout

398 experiment, data collection and management, and manuscript development. All authors

399 reviewed, improved the manuscript and agree to be accountable for the final manuscript.

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Figures

Figure 1

1a) greenish-white owers, Fig 1b) purple-tinted white owers, Fig 1c) white pod Fig 1d) purple tinted- white, Fig 1e) Cream coat seed, Fig 1 f) Black coat seed, Fig 1g) brown coat seed, Fig 1h) buttery shape eyes, Fig 1i) triangular eyes, Fig 1j) irregular eyes. Figure 2 projection of discriminating traits with seed coat color onto the canonical axes 1 and 2. Figure 3

Boxplots showing relationship between ower color and quantitative traits. Figure 4

PCA biplot of quantitative trait for 81 Kersting's groundnut accessions (Axis 1 and 2) Figure 5

PCA biplot of quantitative trait for 81 Kersting's groundnut accessions (Axis 1 and 3) Figure 6

Hierarchical clustering of Kersting's groundnut accessions based on quantitative traits.